Basic Vector Information
Gene/Insert name FnCpf1 (humanized) Alt name type V CRISPR-associated protein Cpf1 (humanized) Alt name Cpf1 (humanized) Alt name Francisella tularensis subsp. novicida U112 Cpf1 (humanized) Species Francisella tularensis subsp. novicida U112 Insert Size (bp) 3910 Promoter T7 Tags / Fusion Proteins 6xHis (N terminal on backbone) MBP (N terminal on insert) TEV site (N terminal on insert) Nucleoplasmin NLS (C terminal on insert) 3xHA (C terminal on insert)
6-His-MBP-TEV-FnCpf1 vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
6-His-MBP-TEV-FnCpf1 vector Sequence
LOCUS 6-His-MBP-TEV-Fn 10685 bp DNA circular SYN 04-JUL-2018 DEFINITION Bacterial expression plasmid for protein purification. ACCESSION . VERSION . KEYWORDS 6-His-MBP-TEV-FnCpf1. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10685) AUTHORS Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F TITLE Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. JOURNAL Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. PUBMED 26422227 REFERENCE 2 (bases 1 to 10685) TITLE Direct Submission REFERENCE 3 (bases 1 to 10685) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..10685 /mol_type="other DNA" /organism="synthetic DNA construct" rep_origin 532..1120 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature complement(1306..1445) /label=bom /note="basis of mobility region from pBR322" CDS complement(1550..1738) /label=rop /note="Rop protein, which maintains plasmids at low copy number" protein_bind complement(2513..2534) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(2550..3629) /label=lacI /note="lac repressor" promoter complement(3630..3707) /label=lacI promoter promoter 4016..4034 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 4035..4059 /label=lac operator /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." RBS 4074..4096 /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4110..4127 /label=6xHis /note="6xHis affinity tag" CDS 4128..5225 /label=MBP /note="maltose binding protein from E. coli" CDS 5244..5261 /label=thrombin site /note="thrombin recognition and cleavage site" CDS 5268..5288 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" CDS 5289..9188 /gene="cas12a" /label=cas12a /note="CRISPR-associated endonuclease Cas12a from Francisella tularensis subsp. novicida (strain U112). Accession#: A0Q7Q2" CDS 9189..9236 /label=nucleoplasmin NLS /note="bipartite nuclear localization signal from nucleoplasmin" CDS 9243..9323 /label=3xHA /note="three tandem HA epitope tags" CDS 9348..9371 /label=FLAG /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" CDS 9372..9416 /label=AviTag(TM) /note="peptide tag that allows for enzymatic biotinylation" CDS 9432..9449 /label=6xHis /note="6xHis affinity tag" terminator 9516..9563 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 9600..9980 /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 10081..10185 /label=AmpR promoter CDS join(10186..10685,1..358) /label=AmpR /note="beta-lactamase"
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